The aim of this project was to analyze the dynamics and binding characteristics of DNA repair proteins during the response of living human cells to radiation-induced DNA damage. In particular, the impact of dense DNA damage after charged particle irradiation was investigated. Different microscopic techniques were used to determine the accumulation kinetics and exchange rates of the repair proteins NBS1, MDC1, ATM, ATR and 53BP1 in irradiation-induced foci. By applying kinetic models, binding constants could be identified. In unirradiated cells, all proteins behaved differently and were less mobile than expected from pure diffusion. Furthermore, all proteins showed function-dependent accumulation kinetics. Binding of 53BP1 to the chromatin surrounding the DNA damage is independent of the damage density. The amount of protein is the kinetically limiting factor. NBS1 binds in two distinct fractions to the damaged DNA in which the tight binding fraction grows with increasing DNA damage density. The amount of ATR which binds to the damaged DNA also increases with the damage density. The highly concentrated and complex DNA damage after charged particle irradiation together with the increased tight binding fraction of NBS1 presumably results in stronger protein activation. Consequently, the creation of binding sites in the damaged area surrounding chromatin is accelerated and MDC1 together with NBS1 is recruited faster. A high damage density also results in the binding of MDC1 not only to the chromatin in the damaged area but also throughout the entire cell nucleus. These results are evidence of an altered cellular response after irradiation with charged particles and point out the specific biological effects of charged particle irradiation.
In a second project, an experimental setup was designed that makes it possible to measure molecular interactions in living cells with high spatial and temporal resolution. This setup will provide insights in the complex molecular network within cells after DNA damage induction. The fluorescence polarization-based FRET method was combined with a spinning disk confocal microscope for the first time. Through a modular design, the system still provides a conventional setup for confocal fluorescence microscopy. The FRET system was validated with blue/yellow and green/red fluorescence reference standards and was used in preliminary biological experiments.